DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility

DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility Jing Jin, Howard Chi Ho Yim, Hsiao Mei Ellie Chang, Yiwei Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2716767/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Infertility is associated with the alteration of the seminal microbiome. However, how the onset of dysbiosis remains controversial and the involvement of host factors remains elusive. This study investigates the alterations of the seminal microbiome in male infertility and examines the association and function of DEFB119, a reproductive-tract-specific host antimicrobial peptide, on the seminal microbiome and male fertility. Results We analyzed the seminal microbiome by 16S rRNA sequencing in 30 individuals with normal sperm parameters and 58 male-factor infertile patients. While we observed comparable genera, diversity and evenness of bacterial communities, a marked decrease in the modularity of the metacommunities was observed in patients with abnormal spermiogram. Analysis of the protein level of DEFB119 by ELISA revealed a marked elevation of DEFB119 in a subpopulation of male-infertile patients. Elevated seminal DEFB119 was associated with a decrease in the observed genera, diversity and evenness of bacterial communities and further distortion of the metacommunities. Mediation analysis suggests the involvement of elevated DEFB119 and dysbiosis of the seminal microbiome in mediating the abnormalities in the spermiogram. Functional experiments showed that recombinant DEFB119 significantly decrease the progressive motility of sperm in patients with abnormal spermiogram. Moreover, DEFB119 demonstrated species-specific antimicrobial activity against common seminal and non-seminal species. Conclusions Male infertility is associated with distorted bacterial networks. Elevation of the DEFB119 level in seminal plasma stratifies male infertile patients with dysbiosis of the seminal microbiome. Both elevated DEFB119 and dysbiosis contribute to the abnormal spermiogram. Our work identifies an important host factor that mediates the host-microbiome interaction and stratifies the seminal microbiome associated with male infertility. These results may lead to a new diagnostic method for male infertility and regimens for formulating the microbiome in the reproductive tract and other organ systems. β-defensin bacterial networks host-microbe interaction microbiota Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background The microbiomes of the reproductive tracts play pivotal roles in fecundity [1–5]. Defects of which represent an important cause of infertility that affects 15% of couples worldwide [6, 7]. The microbiome of the semen herein referred to as the seminal microbiome, has been shown to be modulated by both physiological, environmental and genetic factors [2]. Previous studies have shown that the major bacterial phyla in the seminal microbiome are Firmicutes , Proteobacteria , Actinobacteria , and Bacteroidetes [2] . This balanced composition of the seminal microbiome is regulated by both the innate immune response and secretions by various glands in the male reproductive tract (MRT). Alteration in the healthy composition of the seminal microbiome is associated with sperm abnormalities such as motility and DNA damage [8, 9]. Apart from the involvement in regulating sperm functions and qualities, the seminal microbiome also alters the microenvironment of the female reproductive tract (FRT) after deposition. The seminal microbiome can be shared between heterosexual couples, giving complementary semino-vaginal microbiomes that affect the sperm migration along the FRT, the conception and embryo development in the oviduct, and the receptivity of the uterus for implantation [10, 11]. While several phyla of the seminal microbiome have been shown to be associated with sperm abnormalities, the onset of dysbiosis in infertile cases remains controversial. Moreover, the question as to whether there is a host factor involved in the host-microbes interactions remains unknown. The β-defensin family is a group of small antimicrobial peptides mainly expressed by the epithelial cells [12]. Ubiquitously expressed β-defensins such as HBD1-4 play major roles in host defence and innate immune responses [13]. Intriguingly, a number of β-defensin family members are specifically expressed in the MRT. These reproductive-tract enriched/specific β-defensins play important functions in sperm functions and infertility [14, 15]. Surprisingly, although β-defensins are present in semen and possess antimicrobial activity, their physiological functions and involvement in modulating the seminal microbiome have not been explored. DEFB119 is a reproductive tract-specific β-defensin highly expressed in the testis and the epididymis [16]. Single-cell sequencing analysis of testicular cells isolated from non-obstructive azoospermia men demonstrates an elevated expression of DEFB119 in the Sertoli cells [17]. Male mice lacking the mouse ortholog DEFB19 demonstrated subfertility in both sexes [18, 19]. DEFB119 is also expressed in sperm and is involved in sperm chemotaxis [19]. However, the antimicrobial activity of DEFB119 and its potential involvement in regulating the reproductive tract microbiome has not been reported. In view of the antimicrobial activity conserved among members of the β-defensin family, we hypothesize that reproductive-tract β-defensin serves as a host factor in maintaining the seminal microbiome. Defects of which may lead to abnormal spermiogram and contribute to male infertility. In this study, we investigate the seminal microbiome of infertile couples with normal or abnormal spermiogram parameters. We also investigate the involvement and function of host factor DEFB119 on the dysbiosis of the seminal microbiome. Methods Study design and participants Infertile men aged 25 - 44 years who attended the assisted reproduction clinic at the Prince of Wales Hospital of The Chinese University of Hong Kong were recruited. All participants provided written informed consent and local ethics approvals were obtained for this study (approval number: CREC2016.499). All participants were in good health and none of them was under antibiotic therapy during sampling. Semen samples were collected according to a standardized protocol after 2-7 days of abstinence. Specifically, men were instructed to void, wash their hands with soap and water, and cleanse the glans penis with water before collecting the sample. Samples were collected via masturbation into a sterile container, without the use of saliva or lubrication, and immediately provided to clinical staff for processing and sample storage. For diagnosis of male-factor infertility, semen analysis was performed according to the World Health Organization (WHO) 2010 guidelines [20]. Briefly, semen samples were allowed to liquefy for up to 30 minutes at 37°C. The samples were manually evaluated for volume and then assessed using optical microscopy for concentration, percentage of total motility, percentage of progressive motility, total motile sperm count, and percentage of normal morphology. Infertile couples without a known cause of infertility were included as idiopathic infertility. To examine the association between DEFB119 levels and male infertility, the levels of DEFB119 in seminal plasma were determined by ELISA. Infertile men were categorized according to the level of DEFB119. The seminal microbiome in the categorized patients was determined by 16S rRNA gene sequencing. The relationship between the level of DEFB119 and the phylotypes in the seminal microbiome of participants was analyzed. Immunostaining analysis Sperm smears were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-100, then sperm were blocked with 1% BSA, 22.52 mg/ml glycine in PBST. These sperm were probed with Anti-DEFB119 antibody (ab157790) at 1:100 in PBST with 1% BSA and incubated overnight at 4°C. After washing in PBS, the sperm were incubated with Alexa-fluor 488 conjugated anti-rabbit IgG (ab21206) at 1:500 in PBST at RT in the dark. Slides were mounted with Vecta Mount Mounting Medium (H5000) after washing in PBS. Images were captured with a fluorescent microscope. DEFB119 ELISA analysis Seminal fluid was collected from the supernatant of semen after centrifugation at 2,000g for 15 min. and the assay was conducted according to the manufacturer’s instruction (Human DEFB119 ELISA Kit - LS-F13148). Briefly, The seminal fluid was diluted at 1:100 with sample Diluent, then the mixture was added to a coated Strip Plate and incubated for 2 h at 37°C. After washing, Detection reagent A was added and incubated for 1 hour at 37°C. After washing, Detection reagent B was added and incubated for 1 hr at 37°C. Finally, the TMB substrate was added and incubated for 20 minutes at 37°C in the dark at 37°C. The reaction was then quenched with the Stop solution and the absorbance was measured at 450 nm. The raw data was calculated by the online tool https://elisaanalysis.com. DNA extraction and library preparation Seminal plasma DNA was extracted from the seminal fluid using the QIAamp DNA mini kit (Qiagen#51306). Briefly, A total of 300ul semen was centrifuged at 167,000 g for 15 minutes, 180 ul Buffer ATL and 40 ul proteinase K were added into the pellet and incubated at 56°C overnight with agitation at 700 rpm. Then the Buffer AL was added to the mixture and incubated at 70°C for 30 minutes. The DNA was precipitated with 100% ethanol and loaded into QIAamp Spin Column. After washing with Buffer AW1 and AW2, the DNA was eluted with a total of 40 ul of prewarmed water. DNA concentration and quality were measured by Nanodrop spectrophotometer. We adopted a nested PCR protocol to generate amplicon products (appendix for primer design). First, the full length of the 16S rRNA gene was amplified in primary PCR (20 cycles) using KAPA HiFi HotStart Readymix (Roche). The V3 and V4 hypervariable regions of the 16S rRNA were amplified by internal primers in secondary PCR (25 cycles) using the same reagents. The amplicons were purified by AMPure XP beads to remove unbound primers and dimers according to the manufacturer’s manual. The purified amplicons were attached to dual indices and Illumina sequencing adapters using the Nextera XT Index Kit (FC-131-2001). The indexed library was validated by Qubit™ dsDNA HS Assay Kit (Q32851) and Bioanalyzer Agilent DNA 1000 Kit (5067-1505). Next-generation sequencing The library was sequenced by the Illumina HiSeq system with ~31.93 M paired-end reads. The raw data was processed using a Singularity pipeline developed by the Microbiome Research Centre of The University of New South Wales) that was wrapped with Quantitative Insights into Microbial Ecology (QIIME2, v2020.8) software performed on a high-performance cluster [21]. Overall sequence quality was assessed with Fastp [22], and background noise and chimeric reads were removed with DADA2 [23]. Host decontamination was conducted by mapping the human genome. The amplicon sequence variants (ASVs), which allow precise identification of microbes [24], were aligned to reference sequences at a cutoff of ≥ 99% in Greengenes (v13.5) [25], and normalized using a single rarefaction at 2900 features. Environmental contaminants were identified by the negative controls and removed from subsequent analysis using the Decontam R package. Downstream analysis was completed by the R package microeco (v0.3.2) [26]. Recombinant DEFB119 treatment Recombinant DEFB119 was commercially available (Cloud-Clone Corp, RP1653Hu01). The semen samples were liquefied at room temperature for 30 minutes, and semen analysis was performed as described above. Samples with ≥ 40% total sperm motility (40% were used to examine the effect of rDEFB119 on sperm motility. From each sample, two aliquots of 300 μl raw semen were incubated with vehicle control or 2 μg rDEFB119 at 37°C for 1 hour. Sperm total and progressive motility were manually evaluated after treatment. Bactericidal assay Streptococcus intermedius (JTH08 strain; isolate ID: CC00038), Streptococcus anginosis (isolate ID: CC00108), Streptococcus mitis (ATCC49456 strain; isolate ID: CC00095), Pseudomonas aeruginosa (SNP0614 strain; isolate ID: CC00104), Prevotella stercorea (strain CB35, isolate ID: CC00801), Prevotella copri (strain JCM 13464; isolate ID: CC00987) were kindly provided by Dr. Samuel Forster’s group at Hudson Institute of Medical Research [27]. Streptococcus and Pseudomonas were cultured on blood agar plates and colonies were sub-cultured in BHI broth 24 hours prior to the experiment. Prevotella was cultured on pre-reduced YCFA plates and subcultured in YCFA broth 48 hours prior to the experiment. On the day of the experiment, bacterial culture containing Pseudomonas aeruginosa (1.2-1.4x10 7 CFU), Streptococcus mitis (2-2.2x10 4 CFU), Streptococcus anginosus (0.5-1.4x10 7 CFU), Streptococcus intermedius (2.4-2.8x10 7 CFU), Prevotella stercorea (1.2x10 5 CFU) and Prevotella copri (80 CFU) were mixed with 3-12 ug/ml rDEFB119 or vehicle control in a 96-well plate with a total volume of 200 ul and were incubated under aerobic condition for 24 hours for Pseudomonas or anaerobic conditions for 24 hours for Streptococcus and for 48 hours for Prevotella . Optical density at 620 nm was measured after the incubation. Statistical analysis Statistical analysis of the seminal microbiome was conducted in R 4.0.4. A series of α-diversity analyses were calculated by the Kruskal-Wallis rank-sum test among groups. β-diversity was calculated based on unweighted and weighted UniFrac distance metrics. The PERMANOVA test on β-diversity with 999 permutations was analyzed to compare the significant community dissimilarity among groups. The ANCOM analysis, another test for microbial composition, was performed to test differential bacteria among groups. The differential biomarkers among groups were picked by both the Random forest method and Linear discriminant analysis effect size (LEfSe) method with α equal to 0.05 and an LDA score threshold of 3.0. Demographic characteristics across groups were compared using Mann-Whitney tests (two groups) or Kruskal-Wallis test (three groups) with Dunn’s multiple comparison test for continuous variables and the Chi-square test for categorical variables. Mediation analyses were performed following the published protocol with minor modifications [28]. Briefly, significant mediation effects were assessed between sequential pairs of factors along the microbiome (M)–DEFB119 (D)–spermiogram(S) axis in the normal and infertile groups respectively using R mediate package, with subject age (A) being covariates. Six models of mediation were tested. For example, mediation analysis for Model 1: DEFB119(D)→ microbiome(M, ie. each ASVs)→spermiogram(S, each spermiogram) was performed by fitting into 2 linear models, M =α1+β1D+δ1A+εi1 and S =α2+β2D+γ1M+δ2A+εi2 PThe adjusted P values of the mediation models from Mediate R package were obtained for each model. P-value < 0.05 represents statistical significance. Data availability The 16s rRNA gene sequencing data are available in the Sequence Read Archive database with BioProject ID: PRJNA747100. Pre-publication access for reviewer is available at: https://dataview.ncbi.nlm.nih.gov/object/PRJNA747100?reviewer=vvq8rtf3tn7bisaafjtugpapse Results Distorted bacterial network in male factor infertility To compare the seminal microbiome in men with normal or abnormal spermiogram, we recruited 88 patients seeking assisted reproduction with either abnormal (male-factor infertility, n=58) or normal spermiogram (partners of female-factor infertility or idiopathic infertility, n=30) (Supplemental Table S1). Patients with abnormal spermiogram include oligozoospermia, asthenozoospermia, teratozoospermia or combined cases. The age of the patient, the age of the partner, the treatment outcome and semen volume were comparable. However, the sperm concentration, total motility, progressive motility and morphology were significantly lowered in the male-factor infertile group. We then performed 16S rRNA gene sequencing on seminal plasma samples from this cohort of patients. After sequencing, a sequence curation pipeline optimized for analyses of amplicon libraries was performed for quality control with a low sequencing error rate [29]. In total, 2591 amplicon sequence variants (ASVs) were identified across all the seminal plasma samples. After removing all 178 contaminants in the negative controls, 2413 ASVs were maintained. Consistent with previous reports [2], Firmicutes , Proteobacteria , Actinobacteria and Bacteroidetes were the major phyla in the seminal microbiome that constituted at least 80% of the phyla identified (Fig 1A). We then compared the richness and evenness of the bacterial community using multiple indices. In our cohort, we observed a significant difference in Weighted Unifrac distance between the normozoospermia and male-factor infertile group but no significant difference in other α- and β- diversities between the normozoospermia and male-factor infertile group were observed (Fig 1B-D, Supplemental Fig S1-2). At the genus level, we observed an enrichment of Veillonella , Gardnerella and Lactobacillus in normozoospermia patients. However, the differential abundance of these genera was not statistically significant. To further investigate the structure and interaction of seminal microbial communities, we performed community network analysis using the Weighted Correlation Network Analysis (WGCNA) algorithm [30], which has not been applied in the seminal microbiome. In this analysis, the nodes represent ASVs and the edges that connect these nodes represent correlations between ASVs. Notably, while the number of nodes was comparable, we observed a marked decrease in the number of edges in male-factor infertile patients (Fig 1E). In microbial community networks, the ASVs clustered into independent modules, a property known as modularity, with a small group of ASVs serving as module connectors. We observed the absence of a module hub and the disappearance of peripheral nodes in three phyla, Fusobacteria , TM7 , and Spirochaetes in male-factor infertile patients which lead to the shrinkage of network diameter and heterogeneity (Fig 1E-F). These data suggest that the bacterial network is distorted in patients with abnormal spermiogram despite the comparable richness and evenness of the metacommunities. Elevation of DEFB119 stratifies the dysbiosis in male-factor infertility Previous studies have utilized sperm parameters as stratification factors in the analysis of seminal microbiomes. We speculated that a host factor potentially involved in the host-microbe interactions would provide a better stratification of the seminal microbiome and a higher resolution of the dysbiosis associated with male infertility. Therefore, we explored the involvement of β-defensins in regulating the seminal microbiome, we focused on DEFB119, a β-defensin that plays pivotal roles in sperm production and functions [18, 19]. We determined the protein level of DEFB119 in seminal plasma by enzyme-linked immunoassay with an antibody against the C-terminus of the protein. We observed a range of DEFB119 levels in subjects with normal spermiogram (mean 291.30, CI 210.50 – 372.00 ng/ml). Intriguingly, while the patients with abnormal spermiogram expressed a subtle increased level of DEFB119 in the seminal plasma (mean 393.60, CI 245·70 - 541·50 ng/ml), a subgroup of patients demonstrated a marked elevation of DEFB119 in seminal plasma above the 100th centile (>900 ng/ml) of subjects with normal spermiogram (Fig 2A & Table 1). To examine the metacommunity structure associated with elevated DEFB119, we categorized the patients according to the following grouping: G1 - Normal spermiogram and low DEFB119 level (n=30); G2 - Abnormal spermiogram and low DEFB119 (n=52); and G3 - Abnormal spermiogram and elevated level of DEFB119 (n=5). We observed a lowered sperm concentration, motility and morphology in G2 and G3 as compared to G1. However, the spermiogram parameters were comparable in male-factor infertile patients with normal (G2) or elevated levels of DEFB119 (G3)(Table 1). The phylotypes in G3 were dominated by Firmicutes and Proteobacteria while the abundance of Actinobacteria and Bacteroidetes was diminished (Fig 2B). Comparing the top forty most abundant genera, the abundance of Bactobacillus , Gardnerella , and Prevotella decreased (Fig 2C). We have also compared the richness and evenness of the bacterial community among the three groups using multiple indices as in our previous analysis. The α-diversity was significantly reduced in patients with elevated levels of DEFB119 as compared to G1 and G2 groups (Shannon index p < 0.05, Fig 2D and Supplemental Fig S3). Similarly, the β-diversity as measured by Jaccard distance, Bray Curtis Distance and Weighted Unifrac distance showed that the bacterial community in patients with high levels of DEFB119 (G3) was significantly different from those in G1 and G2 groups (p < 0.05, Fig 2E-F, Supplemental Fig S4). No significant difference in α- and β- diversities except Weighted Unifrac distance were observed between G1 and G2 groups. Differential abundance analysis at the genus level revealed a decrease in Clostridium and increases in eight genera in G3 (p < 0.05), including Sporosarcina , AF12 , Helicobacter , Desulfovibrio , Phyllobacterium , Enterobacter , Anaerobacillus and Carnobacterium , despite their rare occurrence and low relative abundance (Supplemental Fig S5). WGCNA community network analysis showed that patients with elevated levels of DEFB119 (G3) showed a marked decrease in the numbers of both nodes and edges (Fig 2G). The network diameter and heterogeneity were further diminished in G3 as compared to G2. These data suggest that the elevated level of DEFB119 is associated with dysbiosis of the seminal microbiome and severe distortion of bacterial networks in male-factor infertile patients. Mediators of abnormal sperm parameters in male infertility Members of the β-defensin family are known to regulate sperm functions required for the migration in the female reproductive tract and successful fertilization [14, 15]. While our data showed that the elevated level of DEFB119 was associated with the dysbiosis of the seminal microbiome in male infertility, the infertile outcome could be attributed to the effect of DEFB119 on sperm functions per se , the indirect effect from the dysbiosis of the seminal microbiome or both. To study this, we examined the correlation of DEFB119 level with various sperm parameters in normozoospermia and male-factor infertile patients. Among the sperm parameters, a significant negative correlation was observed in progressive motility (Supplemental Fig S6, p < 0.05). In line with this, redundancy analysis (RDA) also revealed a negative correlation of DEFB119 with sperm motility and progressive motility (Fig 3A). Although genera such as Gardnerella and Campylobacter were positively associated with sperm concentration, Veillonella was positively associated with sperm motility and Prevotella was positively associated with sperm morphology and concentration, these associations were not significant (Fig 3A). Next, we set out to test if the abnormal sperm parameter was mediated by the elevation of DEFB119 and the dysbiosis of the seminal microbiome. Mediation analysis revealed a notable increase in the number of significant mediations in the male-factor infertile patients but not the normozoospermia control cohort (Fig 3B). Unexpectedly, significant mediations were observed in 4 out of 6 tested models, regardless of the initiators and mediators. These results suggest that the elevation of DEFB119 and the dysbiosis of the seminal microbiome could be the cause or the effect and vice versa. Nonetheless, the RDA and mediation analysis suggests the possible involvement of elevated DEFB119 and dysbiotic seminal microbiome in mediating the abnormal sperm parameters. Elevated DEFB119 decreases sperm motility in male infertility To validate the mediation analysis, we set out to examine the effect of elevated DEFB119 on sperm parameters. We mimicked the elevated level of DEFB119, as observed in G3, by recombinant DEFB119 (rDEFB119) treatment in a separate cohort of patients with normal or abnormal spermiogram profiles and normal level of DEFB119 in the seminal plasma i.e. being classified as G1 or G2. Since the decrease in sperm count and morphology are spermatogenic factors that would not be altered in a short incubation period of rDEFB119 treatment theoretically and both progressive motility and total motility demonstrated a negative correlation with DEFB119 in RDA (Fig 3A), we examined if the elevated DEFB119 affects the motility of sperm. Our results showed that rDEFB119 significantly decreased both total and progressive motility when compared with the vehicle control (Fig 4A-B, p<0·001). Interestingly, when the sample cohort was further categorized into normozoospermic and male-factor infertile groups based on the spermiogram profile, the decrease in progressive motility was only observed only in male-factor infertile patients (Fig 4C-D). These results, in corroboration with the mediation analysis, suggest that elevated DEFB119 level has detrimental effects on sperm motility, and may contribute to the infertile outcome of the male-factor infertile patients with disturbed seminal microbial networks. Species-specific antimicrobial activity of DEFB119 shapes the seminal microbiome It is well established that the β-defensins possess antimicrobial activity that contributes to the host defence against pathogens [31–35]. Of note, reproductive-tract-specific β-defensins play dual roles in host defence and sperm functions in the reproductive tract, their expressions are known to be induced by both physiological and pathological stimuli including hormone and bacterial toxin lipopolysaccharides [36, 37]. Therefore, the elevated DEFB119 in seminal plasma could be a cause or an effect of the dysbiosis of the seminal microbiome. To investigate if DEFB119 plays an active role in shaping the seminal microbiome and provoking dysbiosis, we performed bactericidal assay against two dominant genera observed in G1 and G2, Prevotella and Streptococcus , and the genus found in G3, Pseudomonas (Fig 5A-C). We include species that can be propagated in vitro and have either been reported in the semen, including Prevotella copri , Streptococcus mitis , Streptococcus anginosus and Pseudomonas aeruginosa [38–41], or found in other organ systems such as Prevotella stercorea in the gut microbiome and Streptococcus intermedius in the central nervous system [42, 43]. We treated the bacterial culture with various doses of rDEFB119 and observed the bacterial growth after culture. We observed a dose-dependent decrease in the amount of Prevotella stercorea , Streptococcus mitis , Streptococcus anginosus and Streptococcus intermedius after rDEFB119 treatments as compared to the vehicle controls, suggesting the bactericidal effect of DEFB119 on these species (Fig 5D-I). Intriguingly, rDEFB119 significantly promoted the growth of Streptococcus mitis at 3 ug/ml while higher doses demonstrate significant inhibitory effects. This result suggests that reproductive tract β-defensins can shape the seminal microbiome by exerting both promoting and inhibitory effects on specific species in a dose-dependent manner. Recombinant DEFB119 exerted a negligible effect on the growth of Pseudomonas aeruginosa and Prevotella copri regardless of the dosage used (Fig 5D-I), suggesting that these bacteria were resistant to DEFB119. Taken together, our results suggest that the elevation of DEFB119 in semen plays ab active role in provoking the dysbiosis of the seminal microbiome. Discussion The present study is the first to characterize host-microbiome interaction in the seminal plasma of infertile patients via a reproductive tract-specific β-defensin. Members of the β-defensin family are involved in host defence and sperm functions, e.g. sperm motility and sperm-egg interaction, in the male and female reproductive tracts. Disorder in these β-defensins expressions, caused either by mutations or decreased expression, is associated with male infertility. Notably, our results showed that an elevated level of DEFB119 was significantly associated with male infertility. More importantly, we revealed a significant correlation between the elevated level of DEFB119 and the decrease in the abundance of genera, diversities and community networks of the seminal microbiome in male-factor infertile patients. In contrast, the elevated level of DEFB119 was not associated with the spermiogram parameters tested. Further functional analysis revealed that elevated DEFB119 decreases the progressive motility of sperm in male-infertile patients but not normozoospermic individuals. These results suggest that both the elevated level of DEFB119 and the dysbiosis of the seminal microbiome contribute to the infertile outcome. It should be noted that the spermiogram analysis does not include a complete profile of sperm functions, particularly those to be triggered in the female reproductive tract. Furthermore, the seminal microbiome can alter the microbiome in the female tract which affects fertilization and embryo development. Therefore, it is possible that the altered seminal microbiome stemming from elevated levels of DEFB119 may lead to deregulation in the event that occurs in the female reproductive tract that contributes to the infertile outcome. Nonetheless, the present study opens a new area of research on the etiology of male infertility attributed to the interplay between β-defensin and the seminal microbiome. Previous studies by several groups have shown that specific phylotypes of the seminal microbiome are positively or negatively associated with abnormal spermiogram i.e. male infertility. For example, the Lactobacillus -predominant phylotype is observed in the normozoospermic patient, Pseudomonas -predominant and Prevotella -predominant phylotype is associated with abnormal spermiogram [8, 41]. In line with these findings, in the subgroup of patients with elevated levels of DEFB119, we also observed a decrease in Lactobacillus and an increase in Pseudomonas . At the species level, we observed both promoting or inhibiting roles of DEFB119 on Streptococcus and Prevotella but not Pseudomonas in a dose-dependent manner, suggesting that DEFB119 provokes the dysbiosis of the seminal microbiome. To this end, it is noteworthy that although we did not identify a phylotype associated with abnormal spermiogram in our cohort of male-factor infertility patients with normal levels of DEFB119, a decrease in the diameter of the microbial community networks and heterogeneity was observed. These findings suggest that a distorted communities network could be an event independent of the elevation of DEFB119. More importantly, dysbiosis was developed in patients with elevated DEFB119 (G3) but not those with normal levels of DEFB119 (G1 and 2). Therefore, our results have identified a previously unknown host factor that stratifies the dysbiosis from distorted microbial networks in male infertility. Further investigation of the stratification by a combination of host factors and sperm parameters will provide a better resolution on the phylotype that leads to the infertile outcome. The human microbiota, particularly those in the gut, oral cavity and skin are known to play important roles in health and disease. The endogenously secreted anti-microbial peptides, including several members of the β-defensin family as well as other defensins, are known to be correlated with unique phylotypes in various organ systems. Our results showed that β-defensin DEFB119 specifically expressed in the reproductive tract caused a significant decrease in Actinobacteria , Bacteroidetes , Tenericutes and Fusobacteria phyla but an increase in Proteobacteria and Firmicutes . The increased phyla demonstrate resistance to the antimicrobial activity of DEFB119. Similar resistance is also observed at the species level against Pseudomonas aeruginosa . Moreover, at the species level, we observed the antimicrobial activity of DEFB119 against Prevotella stercorea and Streptococcus intermedius , both of which have not been found in the semen. These results suggest species-specific antimicrobial activity against bacteria commonly found in the semen, as well as those in other organ systems. In view of the strong expression of a plethora of β-defensin family members in the reproductive tract and the specificity of the antimicrobial activity of individual β-defensin, we postulate that the reproductive tract-specific β-defensins represent a valuable immunocompatible way to formulate desirable microbiome phylotypes in other organ systems so as to restore normal tissue homeostasis. Conclusion Taken together, our data indicate that Male infertility is associated with distorted bacterial networks. The elevation of DEFB119 in seminal plasma lower sperm motility in male infertile patients and provoke the dysbiosis of the seminal microbiome with a decrease in the number of observed genera, diversity, evenness and community networks. Our work has provided novel insight into the host-microbiome interaction via reproductive-tract-specific antimicrobial peptides, which shed light on the etiology of male infertility and may provide a valuable tool for formulating microbiomes in other organ systems. Abbreviations MRT – male reproductive tract DNA – deoxyribonucleic acid FRT – female reproductive tract rRNA – ribosomal ribonucleic acid ELISA – enzyme-linked immunoassay CI – confidence interval ASV – amplicon sequence variant RDA – redundancy analysis WGCNA – Weighted Correlation Network Analysis PCR – polymerase chain reaction Declaraions Ethics approval and consent to participate All participants provided written informed consent and local ethics approvals were obtained for this study. Consent for publication Not applicable. Availability of data and material All sequencing data are available at Sequence Read Archive database. Competing interests The authors declare no conflict of interest. Funding This work was partially funded by Health and Medical Research Fund, Department of Health, Hong Kong SAR government (06170476 to EKLF, 17180701 to MBWC, DYLC, HCHY, EKLF and 06170246 to DYLC) and Lo Kwee Seong Start-up Fund to EKLF. Authors’ contributions HCHY, and EKLF conceived and design the study. JJ, HMEC, CSYC, OAA, and DYLC participated in sample collection the acquisition of data. JJ, HCHY, HMEC, YW, JL, XJ, EKLF analyzed and interpreted the data. JJ, HCHY, and EKLF drafted and revised the manuscript. Acknowledgements We would like to thank the Dr Samuel Forster (Hudson Institute of Medical Research, Australia) and Prof Georgina Hold (University of New South Wales, Australia) for providing the bacterial species. We would also like to thank the core facilities of the School of Biomedical Sciences for providing technical support. References Lundy SD, Vij SC, Rezk AH, Cohen JA, Bajic P, Ramasamy R. The microbiome of the infertile male. Curr Opin Urol. 2020;30:355–362. doi:10.1097/MOU.0000000000000742. Altmäe S, Franasiak JM, Mändar R. The seminal microbiome in health and disease. Nat Rev Urol. 2019;16:703–721. doi:10.1038/s41585-019-0250-y. Koedooder R, Mackens S, Budding A, Fares D, Blockeel C, Laven J, et al. Identification and evaluation of the microbiome in the female and male reproductive tracts. Hum Reprod Update. 2019;25:298–325. doi:10.1093/humupd/dmy048. Lundy SD, Sangwan N, Parekh NV, Selvam MKP, Gupta S, McCaffrey P, et al. Functional and taxonomic dysbiosis of the gut, urine, and semen microbiomes in male infertility. Eur Urol. 2021. doi:10.1016/j.eururo.2021.01.014. Alqawasmeh O, Fok E, Yim H, Li T, Chung J, Chan D. The microbiome and male infertility: looking into the past to move forward. Hum Fertil (Camb). 2022;:1–13. doi:10.1080/14647273.2022.2098540. Agarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37. doi:10.1186/s12958-015-0032-1. Osadchiy V, Mills JN, Mayer EA, Eleswarapu SV. The seminal microbiome and male factor infertility. Curr Sex Health Rep. 2020;12:202–207. doi:10.1007/s11930-020-00273-5. Baud D, Pattaroni C, Vulliemoz N, Castella V, Marsland BJ, Stojanov M. Sperm microbiota and its impact on semen parameters. Front Microbiol. 2019;10:234. doi:10.3389/fmicb.2019.00234. Farahani L, Tharakan T, Yap T, Ramsay JW, Jayasena CN, Minhas S. The semen microbiome and its impact on sperm function and male fertility: A systematic review and meta-analysis. Andrology. 2021;9:115–144. doi:10.1111/andr.12886. Schjenken JE, Robertson SA. The female response to seminal fluid. Physiol Rev. 2020;100:1077–1117. doi:10.1152/physrev.00013.2018. Mändar R, Punab M, Borovkova N, Lapp E, Kiiker R, Korrovits P, et al. Complementary seminovaginal microbiome in couples. Res Microbiol. 2015;166:440–447. doi:10.1016/j.resmic.2015.03.009. Pazgier M, Hoover DM, Yang D, Lu W, Lubkowski J. Human beta-defensins. Cell Mol Life Sci. 2006;63:1294–1313. doi:10.1007/s00018-005-5540-2. Holly MK, Diaz K, Smith JG. Defensins in viral infection and pathogenesis. Annu Rev Virol. 2017;4:369–391. doi:10.1146/annurev-virology-101416-041734. Tollner TL, Venners SA, Hollox EJ, Yudin AI, Liu X, Tang G, et al. A common mutation in the defensin DEFB126 causes impaired sperm function and subfertility. Sci Transl Med. 2011;3:92ra65. doi:10.1126/scitranslmed.3002289. Diao R, Fok KL, Chen H, Yu MK, Duan Y, Chung CM, et al. Deficient human β-defensin 1 underlies male infertility associated with poor sperm motility and genital tract infection. Sci Transl Med. 2014;6:249ra108. doi:10.1126/scitranslmed.3009071. Radhakrishnan Y, Hamil KG, Yenugu S, Young SL, French FS, Hall SH. Identification, characterization, and evolution of a primate beta-defensin gene cluster. Genes Immun. 2005;6:203–210. doi:10.1038/sj.gene.6364184. Wang M, Liu X, Chang G, Chen Y, An G, Yan L, et al. Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis. Cell Stem Cell. 2018;23:599–614.e4. doi:10.1016/j.stem.2018.08.007. Jin J, Li X, Ye M, Qiao F, Chen H, Fok KL. Defb19 regulates the migration of germ cell and is involved in male fertility. Cell Biosci. 2022;12:188. doi:10.1186/s13578-022-00924-1. Li X, Yuan C, Shi J, Kang H, Chen Y, Duan Y, et al. β-Defensin 19/119 mediates sperm chemotaxis and is associated with idiopathic infertility. Cell Rep Med. 2022;:100825. doi:10.1016/j.xcrm.2022.100825. World Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World Health Organization; 2010. Bolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852–857. doi:10.1038/s41587-019-0209-9. Chen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884–i890. doi:10.1093/bioinformatics/bty560. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–583. doi:10.1038/nmeth.3869. Callahan BJ, McMurdie PJ, Holmes SP. Exact sequence variants should replace operational taxonomic units in marker-gene data analysis. ISME J. 2017;11:2639–2643. doi:10.1038/ismej.2017.119. DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006;72:5069–5072. doi:10.1128/AEM.03006-05. Liu C, Cui Y, Li X, Yao M. microeco: an R package for data mining in microbial community ecology. FEMS Microbiol Ecol. 2021;97. doi:10.1093/femsec/fiaa255. Forster SC, Kumar N, Anonye BO, Almeida A, Viciani E, Stares MD, et al. A human gut bacterial genome and culture collection for improved metagenomic analyses. Nat Biotechnol. 2019;37:186–192. doi:10.1038/s41587-018-0009-7. Yan Z, Chen B, Yang Y, Yi X, Wei M, Ecklu-Mensah G, et al. Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions. Nat Microbiol. 2022;7:1361–1375. doi:10.1038/s41564-022-01196-8. Nakatsu G, Li X, Zhou H, Sheng J, Wong SH, Wu WKK, et al. Gut mucosal microbiome across stages of colorectal carcinogenesis. Nat Commun. 2015;6:8727. doi:10.1038/ncomms9727. Langfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559. doi:10.1186/1471-2105-9-559. Schutte BC, McCray PB. [beta]-defensins in lung host defense. Annu Rev Physiol. 2002;64:709–748. doi:10.1146/annurev.physiol.64.081501.134340. Fruitwala S, El-Naccache DW, Chang TL. Multifaceted immune functions of human defensins and underlying mechanisms. Semin Cell Dev Biol. 2019;88:163–172. doi:10.1016/j.semcdb.2018.02.023. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, et al. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 1999;286:525–528. doi:10.1126/science.286.5439.525. Selsted ME, Ouellette AJ. Mammalian defensins in the antimicrobial immune response. Nat Immunol. 2005;6:551–557. doi:10.1038/ni1206. Li P, Chan HC, He B, So SC, Chung YW, Shang Q, et al. An antimicrobial peptide gene found in the male reproductive system of rats. Science. 2001;291:1783–1785. doi:10.1126/science.1056545. Yenugu S, Hamil KG, Radhakrishnan Y, French FS, Hall SH. The androgen-regulated epididymal sperm-binding protein, human beta-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial beta-defensin. Endocrinology. 2004;145:3165–3173. doi:10.1210/en.2003-1698. Contreras G, Shirdel I, Braun MS, Wink M. Defensins: Transcriptional regulation and function beyond antimicrobial activity. Dev Comp Immunol. 2020;104:103556. doi:10.1016/j.dci.2019.103556. Yang H, Zhang J, Xue Z, Zhao C, Lei L, Wen Y, et al. Potential Pathogenic Bacteria in Seminal Microbiota of Patients with Different Types of Dysspermatism. Sci Rep. 2020;10:6876. doi:10.1038/s41598-020-63787-x. Mores CR, Price TK, Wolff B, Halverson T, Limeira R, Brubaker L, et al. Genomic relatedness and clinical significance of Streptococcus mitis strains isolated from the urogenital tract of sexual partners. Microb Genom. 2021;7. doi:10.1099/mgen.0.000535. Moretti E, Capitani S, Figura N, Pammolli A, Federico MG, Giannerini V, et al. The presence of bacteria species in semen and sperm quality. J Assist Reprod Genet. 2009;26:47–56. doi:10.1007/s10815-008-9283-5. Weng S-L, Chiu C-M, Lin F-M, Huang W-C, Liang C, Yang T, et al. Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality. PLoS One. 2014;9:e110152. doi:10.1371/journal.pone.0110152. Yeoh YK, Sun Y, Ip LYT, Wang L, Chan FKL, Miao Y, et al. Prevotella species in the human gut is primarily comprised of Prevotella copri, Prevotella stercorea and related lineages. Sci Rep. 2022;12:9055. doi:10.1038/s41598-022-12721-4. Whiley RA, Beighton D, Winstanley TG, Fraser HY, Hardie JM. Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus (the Streptococcus milleri group): association with different body sites and clinical infections. J Clin Microbiol. 1992;30:243–244. doi:10.1128/jcm.30.1.243-244.1992. Table Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.xlsx Table 1: Clinical characteristics of patients with normozoospermia (G1) and male-factor infertility with (G3) or without (G2) elevation of DEFB119 levels. Data represent mean ± 95% confidence interval. N.A.: Not available SupplementaryinformationEF20230320.pdf Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2716767","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":187044943,"identity":"f5a93b78-63c7-4795-af7a-10fedb26f10a","order_by":0,"name":"Jing Jin","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Jin","suffix":""},{"id":187044944,"identity":"eaa01047-29cb-43e6-a185-bef6da8716e1","order_by":1,"name":"Howard Chi Ho Yim","email":"","orcid":"","institution":"The University of New South Wales","correspondingAuthor":false,"prefix":"","firstName":"Howard","middleName":"Chi Ho","lastName":"Yim","suffix":""},{"id":187044945,"identity":"537eb07e-915a-4d99-9887-9cf168a91907","order_by":2,"name":"Hsiao Mei Ellie Chang","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Hsiao","middleName":"Mei Ellie","lastName":"Chang","suffix":""},{"id":187044946,"identity":"36896484-96a3-48c5-a827-edccd8444497","order_by":3,"name":"Yiwei Wang","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Yiwei","middleName":"","lastName":"Wang","suffix":""},{"id":187044947,"identity":"79d7d488-c376-4fd6-850c-2ffe1517b834","order_by":4,"name":"Sze Yan Chan","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Sze","middleName":"Yan","lastName":"Chan","suffix":""},{"id":187044948,"identity":"6bd64a7d-5cf3-4e99-8cfa-2f3eeb7fc488","order_by":5,"name":"Odai Alqawasmeh","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Odai","middleName":"","lastName":"Alqawasmeh","suffix":""},{"id":187044949,"identity":"41aa4583-0456-49be-8d70-1946fa48325e","order_by":6,"name":"Jinyue Liao","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"Jinyue","middleName":"","lastName":"Liao","suffix":""},{"id":187044950,"identity":"c404cc0b-d1e9-4fa7-b1b5-ceffa9d8c0d3","order_by":7,"name":"Xiaotao Jiang","email":"","orcid":"","institution":"The University of New South Wales","correspondingAuthor":false,"prefix":"","firstName":"Xiaotao","middleName":"","lastName":"Jiang","suffix":""},{"id":187044951,"identity":"66a188cf-c859-41a2-a798-b8bb603a23c3","order_by":8,"name":"David Yiu Leung Chan","email":"","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":false,"prefix":"","firstName":"David","middleName":"Yiu Leung","lastName":"Chan","suffix":""},{"id":187044952,"identity":"8624c838-53cd-40f3-b786-0bbf41081065","order_by":9,"name":"Ellis Kin Lam Fok","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIie3RoQ7CMBCA4WuWgCnMrmLvUHJJMQ28ysiSKTRBjiAwC9jxFlXokhMzewAkPAJuDgo4RAGH6J+c/JK7FiAU+sf6UWlBg4wBeqwEcMMjP4mYIwVIUf5C4EGk/ZbEEVsdu0wjNq261KBTYwckfUSsWUk8K5Rq5+ORgQKNHeaZj0hyhHWk1YkrcQaaGcvR+sjUEbcYaayf5PaZyMeL8YyUTBwxYJ/Eu1hCr1swaYuFqGWOe+LoPT/erOnqXmy029BBVMtJum0qTHzkfU83Hz4yFAqFQl90Byg8Sh/0Yc/nAAAAAElFTkSuQmCC","orcid":"","institution":"The Chinese University of Hong Kong","correspondingAuthor":true,"prefix":"","firstName":"Ellis","middleName":"Kin Lam","lastName":"Fok","suffix":""}],"badges":[],"createdAt":"2023-03-21 05:29:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2716767/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2716767/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":36345836,"identity":"9e4da072-43c1-4d38-9d86-799f352ec21c","added_by":"auto","created_at":"2023-04-26 20:12:04","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1776161,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDistortion of microbial networks in the seminal microbiome of male-factor infertile patients. \u003c/strong\u003e(A) Relative abundance (%) of major phyla in the seminal microbiome of patients with normozoospermia (n=30) and male-factor infertility (n=58). (B) Comparison of α-diversity indices Shannon index between normozoospermia and male-factor infertile patients. (C) Principal Coordinates Analysis of the seminal microbiome of normozoospermia and male-factor infertile patients showing the dissimilarities between groups. (D) Comparison of β-diversity indices Jaccard distance between normozoospermia and male-factor infertile patients. (E) Weighted Correlation Network Analysis (WGCNA) showing the microbial communities networks in the semen of normozoospermia and male-factor infertile patients. Corresponding information is shown in the bottom panel.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/26dfaca84940139a6f20efc5.jpg"},{"id":36345109,"identity":"8519a5d8-9c20-4091-970c-88bfa3b6defb","added_by":"auto","created_at":"2023-04-26 20:04:04","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2024741,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eElevation of DEFB119 is associated with male infertility and dysbiosis of the seminal microbiome. \u003c/strong\u003e(A) Comparison of the protein level of DEFB119 in the seminal plasma collected from normozoospermia and male-factor infertile patients. A threshold of 900 ng/ml was determined to surpass the maximum level observed in normozoospermic subjects. (B) Relative abundance (%) of major phyla in the seminal microbiome of G1 (normozoospermia, normal DEFB119, n=30), G2 (male-infertility, normal DEFB119, n=52) and G3 (male-infertility, elevated DEFB119, n=5). (C) Heat map showing the comparison of the top 40 most abundant genera identified in the three groups of patients. (D) Comparison of α-diversity indices Shannon index between G1, 2 and 3. (E) Principal Coordinates Analysis of G1, 2 and 3 showing the dissimilarities between groups. (F) Comparison of β-diversity indices Jaccard distance between G1, 2 and 3. (G) WGCNA analysis showing the microbial communities networks in G1, 2 and 3. Corresponding information is shown in the bottom panel. Data represent mean ± 95% confidence interval.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/4e29160888196e79e803fbde.jpg"},{"id":36345110,"identity":"b24cdb25-a706-404b-8968-326635e239bd","added_by":"auto","created_at":"2023-04-26 20:04:04","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":584358,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDysbiosis of the seminal microbiome and elevation of DEFB119 are potential mediators of abnormal spermiogram in male infertility. \u003c/strong\u003e(A) Redundancy analysis (RDA) showing the correlation of DEFB119 levels and indicated genera with semen volume and various sperm parameters, including concentration, morphology, motility and progressive motility. (B) Mediation analysis testing the involvement of DEFB119 levels (D) and dysbiosis of the seminal microbiome (M) in mediating the alteration in sperm parameters (S). Two-way ANOVA, *, p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/d94e96201cabbaddef264c32.jpg"},{"id":36345113,"identity":"d9a420e1-bcd6-4144-b82c-384205162f61","added_by":"auto","created_at":"2023-04-26 20:04:04","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":391554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of elevated DEFB119 level on sperm motility. \u003c/strong\u003e(A, B) Sperm total and progressive motility showing the effect of elevated DEFB119 level on sperm from both normozoospermia and male-factor infertile individuals (n=18). (C, D) Sperm progressive motility in normozoospermic (n=6) or male-factor infertile patients (n=12), respectively. *, p\u0026lt;0·05, ***, p\u0026lt;0·001.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/79e4d20bc2e1e304badca6bc.jpg"},{"id":36345838,"identity":"2db1fe82-dd0f-4a86-ba18-bc672a87007b","added_by":"auto","created_at":"2023-04-26 20:12:04","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1053955,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSpecies-specific bactericidal effect of DEFB119. \u003c/strong\u003e(A-C) Relative abundance of the dominant genus, \u003cem\u003ePrevotella\u003c/em\u003e, \u003cem\u003eStreptococcus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e in the semen of G1-3 patients revealed by 16S rRNA sequencing. (D-I) Bactericidal assay of representative species from \u003cem\u003ePseudomonas\u003c/em\u003e (D), \u003cem\u003ePrevotella\u003c/em\u003e (E-F) and \u003cem\u003eStreptococcus\u003c/em\u003e (G-I). Bacterial cultures were treated with the indicated amount of recombinant DEFB119 (rDEFB119) or an equal amount of vehicle control. The percentage change in optical density was calculated with reference to the corresponding 0 ug/ml groups. *, p\u0026lt;0·05, **, p\u0026lt;0·01, ***, p\u0026lt;0·001, ****, p\u0026lt;0·0001.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/77902d3c8f490dd85d214504.jpg"},{"id":38045388,"identity":"1fd8fef2-0600-4e28-8164-a0a7f941a92f","added_by":"auto","created_at":"2023-06-05 17:44:24","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":890107,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/d7f80590-d7e2-4b15-8c01-00e6413bf7cf.pdf"},{"id":36345837,"identity":"dbe44f07-36e6-40cf-9d38-0030281f4911","added_by":"auto","created_at":"2023-04-26 20:12:04","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18416,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTable 1: Clinical characteristics of patients with normozoospermia (G1) and male-factor infertility with (G3) or without (G2) elevation of DEFB119 levels.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData represent mean ± 95% confidence interval. N.A.: Not available\u003c/p\u003e","description":"","filename":"Table1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/980939735b44b4547a4e928d.xlsx"},{"id":36345115,"identity":"76130a29-e407-4d54-a69f-5e0a3877529b","added_by":"auto","created_at":"2023-04-26 20:04:04","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":748925,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryinformationEF20230320.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2716767/v1/ec083a59959835869df4e04f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility","fulltext":[{"header":"Background","content":"\u003cp\u003eThe microbiomes of the reproductive tracts play pivotal roles in fecundity\u0026nbsp;[1\u0026ndash;5]. Defects of which represent an important cause of infertility that affects 15% of couples worldwide\u0026nbsp;[6, 7]. The microbiome of the semen herein referred to as the seminal microbiome, has been shown to be modulated by both physiological, environmental and genetic factors\u0026nbsp;[2]. Previous studies have shown that the major bacterial phyla in the seminal microbiome are \u003cem\u003eFirmicutes\u003c/em\u003e, \u003cem\u003eProteobacteria\u003c/em\u003e, \u003cem\u003eActinobacteria\u003c/em\u003e, and \u003cem\u003eBacteroidetes\u0026nbsp;\u003c/em\u003e\u003cem\u003e[2]\u003c/em\u003e. This balanced composition of the seminal microbiome is regulated by both the innate immune response and secretions by various glands in the male reproductive tract (MRT). Alteration in the healthy composition of the seminal microbiome is associated with sperm abnormalities such as motility and DNA damage\u0026nbsp;[8, 9].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eApart from the involvement in regulating sperm functions and qualities, the seminal microbiome also alters the microenvironment of the female reproductive tract (FRT) after deposition. The seminal microbiome can be shared between heterosexual couples, giving complementary semino-vaginal microbiomes that affect the sperm migration along the FRT, the conception and embryo development in the oviduct, and the receptivity of the uterus for implantation\u0026nbsp;[10, 11]. While several phyla of the seminal microbiome have been shown to be associated with sperm abnormalities, the onset of dysbiosis in infertile cases remains controversial. Moreover, the question as to whether there is a host factor involved in the host-microbes interactions remains unknown.\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe \u0026beta;-defensin family is a group of small antimicrobial peptides mainly expressed by the epithelial cells\u0026nbsp;[12]. Ubiquitously expressed \u0026beta;-defensins such as HBD1-4 play major roles in host defence and innate immune responses\u0026nbsp;[13]. Intriguingly, a number of \u0026beta;-defensin family members are specifically expressed in the MRT. These reproductive-tract enriched/specific \u0026beta;-defensins play important functions in sperm functions and infertility\u0026nbsp;[14, 15]. Surprisingly, although \u0026beta;-defensins are present in semen and possess antimicrobial activity, their physiological functions and involvement in modulating the seminal microbiome have not been explored.\u003c/p\u003e\n\u003cp\u003eDEFB119 is a reproductive tract-specific \u0026beta;-defensin highly expressed in the testis and the epididymis\u0026nbsp;[16]. Single-cell sequencing analysis of testicular cells isolated from non-obstructive azoospermia men demonstrates an elevated expression of DEFB119 in the Sertoli cells\u0026nbsp;[17]. Male mice lacking the mouse ortholog DEFB19 demonstrated subfertility in both sexes\u0026nbsp;[18, 19]. DEFB119 is also expressed in sperm and is involved in sperm chemotaxis\u0026nbsp;[19]. However, the antimicrobial activity of DEFB119 and its potential involvement in regulating the reproductive tract microbiome has not been reported.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn view of the antimicrobial activity conserved among members of the \u0026beta;-defensin family, we hypothesize that reproductive-tract \u0026beta;-defensin serves as a host factor in maintaining the seminal microbiome. Defects of which may lead to abnormal spermiogram and contribute to male infertility. In this study, we investigate the seminal microbiome of infertile couples with normal or abnormal spermiogram parameters. We also investigate the involvement and function of host factor DEFB119 on the dysbiosis of the seminal microbiome.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eStudy design and participants\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eInfertile men aged 25 - 44 years who attended the assisted reproduction clinic at the Prince of Wales Hospital of The Chinese University of Hong Kong were recruited. All participants provided written informed consent and local ethics approvals were obtained for this study (approval number: CREC2016.499). All participants were in good health and none of them was under antibiotic therapy during sampling. Semen samples were collected according to a standardized protocol after 2-7 days of abstinence. Specifically, men were instructed to void, wash their hands with soap and water, and cleanse the glans penis with water before collecting the sample. Samples were collected via masturbation into a sterile container, without the use of saliva or lubrication, and immediately provided to clinical staff for processing and sample storage.\u003c/p\u003e\n\u003cp\u003eFor diagnosis of male-factor infertility, semen analysis was performed according to the World Health Organization (WHO) 2010 guidelines\u0026nbsp;[20]. Briefly, semen samples were allowed to liquefy for up to 30 minutes at 37\u0026deg;C. The samples were manually evaluated for volume and then assessed using optical microscopy for concentration, percentage of total motility, percentage of progressive motility, total motile sperm count, and percentage of normal morphology. Infertile couples without a known cause of infertility were included as idiopathic infertility.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo examine the association between DEFB119 levels and male infertility, the levels of DEFB119 in seminal plasma were determined by ELISA. Infertile men were categorized according to the level of DEFB119. The seminal microbiome in the categorized patients was determined by 16S rRNA gene sequencing. The relationship between the level of DEFB119 and the phylotypes in the seminal microbiome of participants was analyzed. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunostaining analysis\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSperm smears were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton-100, then sperm were blocked with 1% BSA, 22.52 mg/ml glycine in PBST. These sperm were probed with Anti-DEFB119 antibody (ab157790) at 1:100 in PBST with 1% BSA and incubated overnight at 4\u0026deg;C. After washing in PBS, the sperm were incubated with Alexa-fluor 488 conjugated anti-rabbit IgG (ab21206) at 1:500 in PBST at RT in the dark. Slides were mounted with Vecta Mount Mounting Medium (H5000) after washing in PBS. Images were captured with a fluorescent microscope.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDEFB119 ELISA analysis \u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSeminal fluid was collected from the supernatant of semen after centrifugation at 2,000g for 15 min. and the assay was conducted according to the manufacturer\u0026rsquo;s instruction (Human DEFB119 ELISA Kit - LS-F13148). Briefly, The seminal fluid was diluted at 1:100 with sample Diluent, then the mixture was added to a coated Strip Plate and incubated for 2 h at 37\u0026deg;C. After washing, Detection reagent A was added and incubated for 1 hour at 37\u0026deg;C. After washing, Detection reagent B was added and incubated for 1 hr at 37\u0026deg;C. Finally, the TMB substrate was added and incubated for 20 minutes at 37\u0026deg;C in the dark at 37\u0026deg;C. The reaction was then quenched with the Stop solution and the absorbance was measured at 450 nm. The raw data was calculated by the online tool https://elisaanalysis.com.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eDNA extraction and library preparation\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eSeminal plasma DNA was extracted from the seminal fluid using the QIAamp DNA mini kit (Qiagen#51306). Briefly, A total of 300ul semen was centrifuged at 167,000 g for 15 minutes, 180 ul Buffer ATL and 40 ul proteinase K were added into the pellet and incubated at 56\u0026deg;C overnight with agitation at 700 rpm. Then the \u0026nbsp;Buffer AL was added to the mixture and incubated at 70\u0026deg;C for 30 minutes. The DNA was precipitated with 100% ethanol and loaded into QIAamp Spin Column. After washing with Buffer AW1 and AW2, the DNA was eluted with a total of 40 ul of prewarmed water. DNA concentration and quality were measured by Nanodrop spectrophotometer. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe adopted a nested PCR protocol to generate amplicon products (appendix for primer design). First, the full length of the 16S rRNA gene was amplified in primary PCR (20 cycles) using KAPA HiFi HotStart Readymix (Roche). The V3 and V4 hypervariable regions of the 16S rRNA were amplified by internal primers in secondary PCR (25 cycles) using the same reagents. The amplicons were purified by AMPure XP beads to remove unbound primers and dimers according to the manufacturer\u0026rsquo;s manual. The purified amplicons were attached to dual indices and Illumina sequencing adapters using the Nextera XT Index Kit (FC-131-2001). The indexed library was validated by Qubit\u0026trade; dsDNA HS Assay Kit (Q32851) and Bioanalyzer Agilent DNA 1000 Kit (5067-1505). \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eNext-generation sequencing\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe library was sequenced by the Illumina HiSeq system with ~31.93 M paired-end reads. The raw data was processed using a Singularity pipeline developed by the Microbiome Research Centre of The University of New South Wales) that was wrapped with Quantitative Insights into Microbial Ecology (QIIME2, v2020.8) software performed on a high-performance cluster\u0026nbsp;[21]. Overall sequence quality was assessed with Fastp\u0026nbsp;[22], and background noise and chimeric reads were removed with DADA2\u0026nbsp;[23]. Host decontamination was conducted by mapping the human genome. The amplicon sequence variants (ASVs), which allow precise identification of microbes\u0026nbsp;[24], were aligned to reference sequences at a cutoff of \u0026ge; 99% in Greengenes (v13.5)\u0026nbsp;[25], and normalized using a single rarefaction at 2900 features. Environmental contaminants were identified by the negative controls and removed from subsequent analysis using the Decontam R package. Downstream analysis was completed by the R package microeco (v0.3.2)\u0026nbsp;[26].\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eRecombinant DEFB119 treatment\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRecombinant DEFB119 was commercially available (Cloud-Clone Corp, RP1653Hu01). The semen samples were liquefied at room temperature for 30 minutes, and semen analysis was performed as described above. Samples with\u0026nbsp;\u0026ge;\u0026nbsp;40% total sperm motility (40% were used to examine the effect of rDEFB119 on sperm motility. From each sample, two aliquots of 300 \u0026mu;l raw semen were incubated with vehicle control or 2 \u0026mu;g rDEFB119 at 37\u0026deg;C for 1 hour. Sperm total and progressive motility were manually evaluated after treatment.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eBactericidal assay\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStreptococcus intermedius\u003c/em\u003e (JTH08 strain; isolate ID: CC00038), \u003cem\u003eStreptococcus anginosis\u0026nbsp;\u003c/em\u003e(isolate ID: CC00108), \u003cem\u003eStreptococcus mitis\u003c/em\u003e (ATCC49456 strain; isolate ID: CC00095), \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (SNP0614 strain; isolate ID: CC00104), \u003cem\u003ePrevotella stercorea\u0026nbsp;\u003c/em\u003e(strain CB35, isolate ID: CC00801), \u003cem\u003ePrevotella copri\u003c/em\u003e (strain JCM 13464; isolate ID: CC00987) were kindly provided by Dr. Samuel Forster\u0026rsquo;s group at Hudson Institute of Medical Research\u0026nbsp;[27]. \u003cem\u003eStreptococcus\u003c/em\u003e and \u003cem\u003ePseudomonas\u003c/em\u003e were cultured on blood agar plates and colonies were sub-cultured in BHI broth 24 hours prior to the experiment. \u003cem\u003ePrevotella\u003c/em\u003e was cultured on pre-reduced YCFA plates and subcultured in YCFA broth 48 hours prior to the experiment. On the day of the experiment, bacterial culture containing \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e (1.2-1.4x10\u003csup\u003e7\u003c/sup\u003e CFU), \u003cem\u003eStreptococcus mitis\u003c/em\u003e (2-2.2x10\u003csup\u003e4\u003c/sup\u003e CFU), \u003cem\u003eStreptococcus anginosus\u003c/em\u003e (0.5-1.4x10\u003csup\u003e7\u003c/sup\u003e CFU), \u003cem\u003eStreptococcus intermedius\u003c/em\u003e (2.4-2.8x10\u003csup\u003e7\u003c/sup\u003e CFU), \u003cem\u003ePrevotella stercorea\u003c/em\u003e (1.2x10\u003csup\u003e5\u003c/sup\u003e CFU) and \u003cem\u003ePrevotella copri\u003c/em\u003e (80 CFU) were mixed with 3-12 ug/ml rDEFB119 or vehicle control in a 96-well plate with a total volume of 200 ul and were incubated under aerobic condition for 24 hours for \u003cem\u003ePseudomonas\u003c/em\u003e or anaerobic conditions for 24 hours for \u003cem\u003eStreptococcus\u003c/em\u003e and for 48 hours for \u003cem\u003ePrevotella\u003c/em\u003e. Optical density at 620 nm was measured after the incubation. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eStatistical analysis\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eStatistical analysis of the seminal microbiome was conducted in R 4.0.4. A series of \u0026alpha;-diversity analyses were calculated by the Kruskal-Wallis rank-sum test among groups. \u0026beta;-diversity was calculated based on unweighted and weighted UniFrac distance metrics. The PERMANOVA test on \u0026beta;-diversity with 999 permutations was analyzed to compare the significant community dissimilarity among groups. The ANCOM analysis, another test for microbial composition, was performed to test differential bacteria among groups. The differential biomarkers among groups were picked by both the Random forest method and Linear discriminant analysis effect size (LEfSe) method with \u0026alpha; equal to 0.05 and an LDA score threshold of 3.0. Demographic characteristics across groups were compared using Mann-Whitney tests (two groups) or Kruskal-Wallis test (three groups) with Dunn\u0026rsquo;s multiple comparison test for continuous variables and the Chi-square test for categorical variables. Mediation analyses were performed following the published protocol with minor modifications\u0026nbsp;[28]. Briefly, significant mediation effects were assessed between sequential pairs of factors along the microbiome (M)\u0026ndash;DEFB119 (D)\u0026ndash;spermiogram(S) axis in the normal and infertile groups respectively using R mediate package, with subject age (A) being covariates. Six models of mediation were tested. For example, mediation analysis for Model 1: DEFB119(D)\u0026rarr; microbiome(M, ie. each ASVs)\u0026rarr;spermiogram(S, each spermiogram) was performed by fitting into 2 linear models, M =\u0026alpha;1+\u0026beta;1D+\u0026delta;1A+\u0026epsilon;i1 \u0026nbsp;and \u0026nbsp;S =\u0026alpha;2+\u0026beta;2D+\u0026gamma;1M+\u0026delta;2A+\u0026epsilon;i2 \u0026nbsp;PThe adjusted P values of the mediation models from Mediate R package were obtained for each model. P-value \u0026lt; 0.05 represents statistical significance.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eData availability\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe 16s rRNA gene sequencing data are available in the Sequence Read Archive database with BioProject ID: PRJNA747100. Pre-publication access for reviewer is available at: https://dataview.ncbi.nlm.nih.gov/object/PRJNA747100?reviewer=vvq8rtf3tn7bisaafjtugpapse\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003eDistorted bacterial network in male factor infertility\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo compare the seminal microbiome in men with normal or abnormal spermiogram, we recruited 88 patients seeking assisted reproduction with either abnormal (male-factor infertility, n=58) or normal spermiogram (partners of female-factor infertility or idiopathic infertility, n=30) (Supplemental Table S1). Patients with abnormal spermiogram include oligozoospermia, asthenozoospermia, teratozoospermia or combined cases. The age of the patient, the age of the partner, the treatment outcome and semen volume were comparable. However, the sperm concentration, total motility, progressive motility and morphology were significantly lowered in the male-factor infertile group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe then performed 16S rRNA gene sequencing on seminal plasma samples from this cohort of patients. After sequencing, a sequence curation pipeline optimized for analyses of amplicon libraries was performed for quality control with a low sequencing error rate\u0026nbsp;[29]. In total, 2591 amplicon sequence variants (ASVs) were identified across all the seminal plasma samples. After removing all 178 contaminants in the negative controls, 2413 ASVs were maintained. Consistent with previous reports\u0026nbsp;[2], \u003cem\u003eFirmicutes\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Proteobacteria\u003c/em\u003e,\u003cem\u003e\u0026nbsp;Actinobacteria\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Bacteroidetes\u0026nbsp;\u003c/em\u003ewere the major phyla in the seminal microbiome that constituted at least 80% of the phyla identified (Fig 1A). We then compared the richness and evenness of the bacterial community using multiple indices. In our cohort, we observed a significant difference in Weighted Unifrac distance between the normozoospermia and male-factor infertile group but no significant difference in other \u0026alpha;- and \u0026beta;- diversities between the normozoospermia and male-factor infertile group were observed (Fig 1B-D, Supplemental Fig S1-2). At the genus level, we observed an enrichment of \u003cem\u003eVeillonella\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e and \u003cem\u003eLactobacillus\u003c/em\u003e in normozoospermia patients. However, the differential abundance of these genera was not statistically significant. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo further investigate the structure and interaction of seminal microbial communities, we performed community network analysis using the Weighted Correlation Network Analysis (WGCNA) algorithm\u0026nbsp;[30], which has not been applied in the seminal microbiome. In this analysis, the nodes represent ASVs and the edges that connect these nodes represent correlations between ASVs. Notably, while the number of nodes was comparable, we observed a marked decrease in the number of edges in male-factor infertile patients (Fig 1E). In microbial community networks, the ASVs clustered into independent modules, a property known as modularity, with a small group of ASVs serving as module connectors. We observed the absence of a module hub and the disappearance of peripheral nodes in three phyla, \u003cem\u003eFusobacteria\u003c/em\u003e, \u0026nbsp;\u003cem\u003eTM7\u003c/em\u003e, and \u003cem\u003eSpirochaetes\u003c/em\u003e in male-factor infertile patients which lead to the shrinkage of network diameter and heterogeneity (Fig 1E-F). \u0026nbsp;These data suggest that the bacterial network is distorted in patients with abnormal spermiogram despite the comparable richness and evenness of the metacommunities. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElevation of DEFB119 stratifies the dysbiosis in male-factor infertility\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePrevious studies have utilized sperm parameters as stratification factors in the analysis of seminal microbiomes. We speculated that a host factor potentially involved in the host-microbe interactions would provide a better stratification of the seminal microbiome and a higher resolution of the dysbiosis associated with male infertility. Therefore, we explored the involvement of \u0026beta;-defensins in regulating the seminal microbiome, we focused on DEFB119, a \u0026beta;-defensin that plays pivotal roles in sperm production and functions\u0026nbsp;[18, 19]. We determined the protein level of DEFB119 in seminal plasma by enzyme-linked immunoassay with an antibody against the C-terminus of the protein. We observed a range of DEFB119 levels in subjects with normal spermiogram (mean 291.30, CI 210.50 \u0026ndash; 372.00 ng/ml). Intriguingly, while the patients with abnormal spermiogram expressed a subtle increased level of DEFB119 in the seminal plasma (mean 393.60, CI 245\u0026middot;70 - 541\u0026middot;50 ng/ml), a subgroup of patients demonstrated a marked elevation of DEFB119 in seminal plasma above the 100th centile (\u0026gt;900 ng/ml) of subjects with normal spermiogram (Fig 2A \u0026amp; Table 1).\u003c/p\u003e\n\u003cp\u003eTo examine the metacommunity structure associated with elevated DEFB119, we categorized the patients according to the following grouping: G1 - Normal spermiogram and low DEFB119 level (n=30); G2 - Abnormal spermiogram and low DEFB119 (n=52); and G3 - Abnormal spermiogram and elevated level of DEFB119 (n=5). We observed a lowered sperm concentration, motility and morphology in G2 and G3 as compared to G1. However, the spermiogram parameters were comparable in male-factor infertile patients with normal (G2) or elevated levels of DEFB119 (G3)(Table 1). The phylotypes in G3 were dominated by \u003cem\u003eFirmicutes\u0026nbsp;\u003c/em\u003eand \u003cem\u003eProteobacteria\u0026nbsp;\u003c/em\u003ewhile the abundance of \u003cem\u003eActinobacteria\u0026nbsp;\u003c/em\u003eand \u003cem\u003eBacteroidetes\u0026nbsp;\u003c/em\u003ewas diminished (Fig 2B). Comparing the top forty most abundant genera, the abundance of \u003cem\u003eBactobacillus\u003c/em\u003e, \u003cem\u003eGardnerella\u003c/em\u003e, and \u003cem\u003ePrevotella\u003c/em\u003e decreased (Fig 2C). We have also compared the richness and evenness of the bacterial community among the three groups using multiple indices as in our previous analysis. The \u0026alpha;-diversity was significantly reduced in patients with elevated levels of DEFB119 as compared to G1 and G2 groups (Shannon index p \u0026lt; 0.05, Fig 2D and Supplemental Fig S3). Similarly, the \u0026beta;-diversity as measured by Jaccard distance, Bray Curtis Distance and Weighted Unifrac distance showed that the bacterial community in patients with high levels of DEFB119 (G3) was significantly different from those in G1 and G2 groups (p \u0026lt; 0.05, Fig 2E-F, Supplemental Fig S4). No significant difference in \u0026alpha;- and \u0026beta;- diversities except Weighted Unifrac distance were observed between G1 and G2 groups.\u003c/p\u003e\n\u003cp\u003eDifferential abundance analysis at the genus level revealed a decrease in Clostridium and increases in eight genera in G3 (p \u0026lt; 0.05), including \u003cem\u003eSporosarcina\u003c/em\u003e, \u003cem\u003eAF12\u003c/em\u003e, \u003cem\u003eHelicobacter\u003c/em\u003e, \u003cem\u003eDesulfovibrio\u003c/em\u003e, \u003cem\u003ePhyllobacterium\u003c/em\u003e, \u003cem\u003eEnterobacter\u003c/em\u003e, \u003cem\u003eAnaerobacillus\u003c/em\u003e and \u003cem\u003eCarnobacterium\u003c/em\u003e, despite their rare occurrence and low relative abundance\u003cem\u003e\u0026nbsp;\u003c/em\u003e(Supplemental Fig S5). WGCNA community network analysis showed that patients with elevated levels of DEFB119 (G3) showed a marked decrease in the numbers of both nodes and edges (Fig 2G). The network diameter and heterogeneity were further diminished in G3 as compared to G2. These data suggest that the elevated level of DEFB119 is associated with dysbiosis of the seminal microbiome and severe distortion of bacterial networks in male-factor infertile patients. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eMediators of abnormal sperm parameters in male infertility\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eMembers of the \u0026beta;-defensin family are known to regulate sperm functions required for the migration in the female reproductive tract and successful fertilization\u0026nbsp;[14, 15]. While our data showed that the elevated level of DEFB119 was associated with the dysbiosis of the seminal microbiome in male infertility, the infertile outcome could be attributed to the effect of DEFB119 on sperm functions \u003cem\u003eper se\u003c/em\u003e, the indirect effect from the dysbiosis of the seminal microbiome or both. To study this, we examined the correlation of DEFB119 level with various sperm parameters in normozoospermia and male-factor infertile patients. Among the sperm parameters, a significant negative correlation was observed in progressive motility (Supplemental Fig S6, p \u0026lt; 0.05). In line with this, redundancy analysis (RDA) also revealed a negative correlation of DEFB119 with sperm motility and progressive motility (Fig 3A). Although genera such as \u003cem\u003eGardnerella\u003c/em\u003e and \u003cem\u003eCampylobacter\u003c/em\u003e were positively associated with sperm concentration, \u003cem\u003eVeillonella\u003c/em\u003e was positively associated with sperm motility and \u003cem\u003ePrevotella\u003c/em\u003e was positively associated with sperm morphology and concentration, these associations were not significant (Fig 3A). Next, we set out to test if the abnormal sperm parameter was mediated by the elevation of DEFB119 and the dysbiosis of the seminal microbiome. Mediation analysis revealed a notable increase in the number of significant mediations in the male-factor infertile patients but not the normozoospermia control cohort (Fig 3B). Unexpectedly, significant mediations were observed in 4 out of 6 tested models, regardless of the initiators and mediators. These results suggest that the elevation of DEFB119 and the dysbiosis of the seminal microbiome could be the cause or the effect and vice versa. Nonetheless, the RDA and mediation analysis suggests the possible involvement of elevated DEFB119 and dysbiotic seminal microbiome in mediating the abnormal sperm parameters. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElevated DEFB119 decreases sperm motility in male infertility\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the mediation analysis, we set out to examine the effect of elevated DEFB119 on sperm parameters. We mimicked the elevated level of DEFB119, as observed in G3, by recombinant DEFB119 (rDEFB119) treatment in a separate cohort of patients with normal or abnormal spermiogram profiles and normal level of DEFB119 in the seminal plasma i.e. being classified as G1 or G2. Since the decrease in sperm count and morphology are spermatogenic factors that would not be altered in a short incubation period of rDEFB119 treatment theoretically and both progressive motility and total motility demonstrated a negative correlation with DEFB119 in RDA (Fig 3A), we examined if the elevated DEFB119 affects the motility of sperm. Our results showed that rDEFB119 significantly decreased both total and progressive motility when compared with the vehicle control (Fig 4A-B, p\u0026lt;0\u0026middot;001). Interestingly, when the sample cohort was further categorized into normozoospermic and male-factor infertile groups based on the spermiogram profile, the decrease in progressive motility was only observed only in male-factor infertile patients (Fig 4C-D). These results, in corroboration with the mediation analysis, suggest that elevated DEFB119 level has detrimental effects on sperm motility, and may contribute to the infertile outcome of the male-factor infertile patients with disturbed seminal microbial networks.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSpecies-specific antimicrobial activity of DEFB119 shapes the seminal microbiome\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIt is well established that the \u0026beta;-defensins possess antimicrobial activity that contributes to the host defence against pathogens\u0026nbsp;[31\u0026ndash;35]. Of note, reproductive-tract-specific \u0026beta;-defensins play dual roles in host defence and sperm functions in the reproductive tract, their expressions are known to be induced by both physiological and pathological stimuli including hormone and bacterial toxin lipopolysaccharides\u0026nbsp;[36, 37]. Therefore, the elevated DEFB119 in seminal plasma could be a cause or an effect of the dysbiosis of the seminal microbiome. To investigate if DEFB119 plays an active role in shaping the seminal microbiome and provoking dysbiosis, we performed bactericidal assay against two dominant genera observed in G1 and G2, \u003cem\u003ePrevotella\u003c/em\u003e and \u003cem\u003eStreptococcus\u003c/em\u003e, and the genus found in G3, \u003cem\u003ePseudomonas\u003c/em\u003e (Fig 5A-C). We include species that can be propagated \u003cem\u003ein vitro\u003c/em\u003e and have either been reported in the semen, including \u003cem\u003ePrevotella copri\u003c/em\u003e, \u003cem\u003eStreptococcus mitis\u003c/em\u003e, \u003cem\u003eStreptococcus anginosus\u003c/em\u003e and \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e [38\u0026ndash;41], or found in other organ systems such as \u003cem\u003ePrevotella stercorea\u003c/em\u003e in the gut microbiome and \u003cem\u003eStreptococcus intermedius\u003c/em\u003e in the central nervous system\u0026nbsp;[42, 43].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe treated the bacterial culture with various doses of rDEFB119 and observed the bacterial growth after culture. We observed a dose-dependent decrease in the amount of \u003cem\u003ePrevotella stercorea\u003c/em\u003e, \u003cem\u003eStreptococcus mitis\u003c/em\u003e, \u003cem\u003eStreptococcus anginosus\u003c/em\u003e and \u003cem\u003eStreptococcus intermedius\u003c/em\u003e after rDEFB119 treatments as compared to the vehicle controls, suggesting the bactericidal effect of DEFB119 on these species (Fig 5D-I). Intriguingly, rDEFB119 significantly promoted the growth of \u003cem\u003eStreptococcus mitis\u003c/em\u003e at 3 ug/ml while higher doses demonstrate significant inhibitory effects. This result suggests that reproductive tract \u0026beta;-defensins can shape the seminal microbiome by exerting both promoting and inhibitory effects on specific species in a dose-dependent manner. Recombinant DEFB119 exerted a negligible effect on the growth of \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e and \u003cem\u003ePrevotella copri\u0026nbsp;\u003c/em\u003eregardless of the dosage used (Fig 5D-I), suggesting that these bacteria were resistant to DEFB119. Taken together, our results suggest that the elevation of DEFB119 in semen plays ab active role in provoking the dysbiosis of the seminal microbiome. \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study is the first to characterize host-microbiome interaction in the seminal plasma of infertile patients via a reproductive tract-specific \u0026beta;-defensin. Members of the \u0026beta;-defensin family are involved in host defence and sperm functions, e.g. sperm motility and sperm-egg interaction, in the male and female reproductive tracts. Disorder in these \u0026beta;-defensins expressions, caused either by mutations or decreased expression, is associated with male infertility. Notably, our results showed that an elevated level of DEFB119 was significantly associated with male infertility. More importantly, we revealed a significant correlation between the elevated level of DEFB119 and the decrease in the abundance of genera, diversities and community networks of the seminal microbiome in male-factor infertile patients. In contrast, the elevated level of DEFB119 was not associated with the spermiogram parameters tested. Further functional analysis revealed that elevated DEFB119 decreases the progressive motility of sperm in male-infertile patients but not normozoospermic individuals. These results suggest that both the elevated level of DEFB119 and the dysbiosis of the seminal microbiome contribute to the infertile outcome. It should be noted that the spermiogram analysis does not include a complete profile of sperm functions, particularly those to be triggered in the female reproductive tract. Furthermore, the seminal microbiome can alter the microbiome in the female tract which affects fertilization and embryo development. Therefore, it is possible that the altered seminal microbiome stemming from elevated levels of DEFB119 may lead to deregulation in the event that occurs in the female reproductive tract that contributes to the infertile outcome. Nonetheless, the present study opens a new area of research on the etiology of male infertility attributed to the interplay between \u0026beta;-defensin and the seminal microbiome.\u003c/p\u003e\n\u003cp\u003ePrevious studies by several groups have shown that specific phylotypes of the seminal microbiome are positively or negatively associated with abnormal spermiogram i.e. male infertility. For example, the \u003cem\u003eLactobacillus\u003c/em\u003e-predominant phylotype is observed in the normozoospermic patient, \u003cem\u003ePseudomonas\u003c/em\u003e-predominant and \u003cem\u003ePrevotella\u003c/em\u003e-predominant phylotype is associated with abnormal spermiogram\u0026nbsp;[8, 41]. In line with these findings, in the subgroup of patients with elevated levels of DEFB119, we also observed a decrease in \u003cem\u003eLactobacillus\u0026nbsp;\u003c/em\u003eand an increase in \u003cem\u003ePseudomonas\u003c/em\u003e. At the species level, we observed both promoting or inhibiting roles of DEFB119 on \u003cem\u003eStreptococcus\u003c/em\u003e and \u003cem\u003ePrevotella\u003c/em\u003e but not \u003cem\u003ePseudomonas\u003c/em\u003e in a dose-dependent manner, suggesting that DEFB119 provokes the dysbiosis of the seminal microbiome. To this end, it is noteworthy that although we did not identify a phylotype associated with abnormal spermiogram in our cohort of male-factor infertility patients with normal levels of DEFB119, a decrease in the diameter of the microbial community networks and heterogeneity was observed. These findings suggest that a distorted communities network could be an event independent of the elevation of DEFB119. More importantly, dysbiosis was developed in patients with elevated DEFB119 (G3) but not those with normal levels of DEFB119 (G1 and 2). Therefore, our results have identified a previously unknown host factor that stratifies the dysbiosis from distorted microbial networks in male infertility. Further investigation of the stratification by a combination of host factors and sperm parameters will provide a better resolution on the phylotype that leads to the infertile outcome.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe human microbiota, particularly those in the gut, oral cavity and skin are known to play important roles in health and disease. The endogenously secreted anti-microbial peptides, including several members of the \u0026beta;-defensin family as well as other defensins, are known to be correlated with unique phylotypes in various organ systems. Our results showed that \u0026beta;-defensin DEFB119 specifically expressed in the reproductive tract caused a significant decrease in \u003cem\u003eActinobacteria\u003c/em\u003e, \u003cem\u003eBacteroidetes\u003c/em\u003e, \u003cem\u003eTenericutes\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFusobacteria\u0026nbsp;\u003c/em\u003ephyla but an increase in \u003cem\u003eProteobacteria\u0026nbsp;\u003c/em\u003eand \u003cem\u003eFirmicutes\u003c/em\u003e. The increased phyla demonstrate resistance to the antimicrobial activity of DEFB119. Similar resistance is also observed at the species level against \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e. Moreover, at the species level, we observed the antimicrobial activity of DEFB119 against \u003cem\u003ePrevotella stercorea\u0026nbsp;\u003c/em\u003eand \u003cem\u003eStreptococcus intermedius\u003c/em\u003e, both of which have not been found in the semen. These results suggest species-specific antimicrobial activity against bacteria commonly found in the semen, as well as those in other organ systems. In view of the strong expression of a plethora of \u0026beta;-defensin family members in the reproductive tract and the specificity of the antimicrobial activity of individual \u0026beta;-defensin, we postulate that the reproductive tract-specific \u0026beta;-defensins represent a valuable immunocompatible way to formulate desirable microbiome phylotypes in other organ systems so as to restore normal tissue homeostasis.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTaken together, our data indicate that Male infertility is associated with distorted bacterial networks. The elevation of DEFB119 in seminal plasma lower sperm motility in male infertile patients and provoke the dysbiosis of the seminal microbiome with a decrease in the number of observed genera, diversity, evenness and community networks. Our work has provided novel insight into the host-microbiome interaction via reproductive-tract-specific antimicrobial peptides, which shed light on the etiology of male infertility and may provide a valuable tool for formulating microbiomes in other organ systems.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eMRT \u0026ndash; male reproductive tract\u003c/p\u003e\n\u003cp\u003eDNA \u0026ndash; deoxyribonucleic acid\u003c/p\u003e\n\u003cp\u003eFRT \u0026ndash; female reproductive tract\u003c/p\u003e\n\u003cp\u003erRNA \u0026ndash; ribosomal ribonucleic acid\u003c/p\u003e\n\u003cp\u003eELISA \u0026ndash; enzyme-linked immunoassay\u003c/p\u003e\n\u003cp\u003eCI \u0026ndash; confidence interval\u003c/p\u003e\n\u003cp\u003eASV \u0026ndash; amplicon sequence variant\u003c/p\u003e\n\u003cp\u003eRDA \u0026ndash; redundancy analysis\u003c/p\u003e\n\u003cp\u003eWGCNA \u0026ndash; Weighted Correlation Network Analysis\u003c/p\u003e\n\u003cp\u003ePCR \u0026ndash; polymerase chain reaction\u003c/p\u003e"},{"header":"Declaraions","content":"\u003cp\u003e\u003cem\u003eEthics approval and consent to participate\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll participants provided written informed consent and local ethics approvals were obtained for this study.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsent for publication\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAvailability of data and material\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAll sequencing data are available at Sequence Read Archive database.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCompeting interests\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eFunding\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially funded by Health and Medical Research Fund, Department of Health, Hong Kong SAR government (06170476 to EKLF, 17180701 to MBWC, DYLC, HCHY, EKLF and 06170246 to DYLC) and Lo Kwee Seong Start-up Fund to EKLF.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAuthors\u0026rsquo; contributions\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eHCHY, and EKLF conceived and design the study. JJ, HMEC, CSYC, OAA, and DYLC participated in sample collection the acquisition of data. JJ, HCHY, HMEC, YW, JL, XJ, EKLF analyzed and interpreted the data. JJ, HCHY, and EKLF drafted and revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAcknowledgements\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Dr Samuel Forster (Hudson Institute of Medical Research, Australia) and Prof Georgina Hold (University of New South Wales, Australia) for providing the bacterial species. We would also like to thank the core facilities of the School of Biomedical Sciences for providing technical support.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLundy SD, Vij SC, Rezk AH, Cohen JA, Bajic P, Ramasamy R. The microbiome of the infertile male. Curr Opin Urol. 2020;30:355\u0026ndash;362. doi:10.1097/MOU.0000000000000742.\u003c/li\u003e\n\u003cli\u003eAltm\u0026auml;e S, Franasiak JM, M\u0026auml;ndar R. The seminal microbiome in health and disease. Nat Rev Urol. 2019;16:703\u0026ndash;721. doi:10.1038/s41585-019-0250-y.\u003c/li\u003e\n\u003cli\u003eKoedooder R, Mackens S, Budding A, Fares D, Blockeel C, Laven J, et al. Identification and evaluation of the microbiome in the female and male reproductive tracts. Hum Reprod Update. 2019;25:298\u0026ndash;325. doi:10.1093/humupd/dmy048.\u003c/li\u003e\n\u003cli\u003eLundy SD, Sangwan N, Parekh NV, Selvam MKP, Gupta S, McCaffrey P, et al. Functional and taxonomic dysbiosis of the gut, urine, and semen microbiomes in male infertility. Eur Urol. 2021. doi:10.1016/j.eururo.2021.01.014.\u003c/li\u003e\n\u003cli\u003eAlqawasmeh O, Fok E, Yim H, Li T, Chung J, Chan D. The microbiome and male infertility: looking into the past to move forward. Hum Fertil (Camb). 2022;:1\u0026ndash;13. doi:10.1080/14647273.2022.2098540.\u003c/li\u003e\n\u003cli\u003eAgarwal A, Mulgund A, Hamada A, Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol. 2015;13:37. doi:10.1186/s12958-015-0032-1.\u003c/li\u003e\n\u003cli\u003eOsadchiy V, Mills JN, Mayer EA, Eleswarapu SV. The seminal microbiome and male factor infertility. Curr Sex Health Rep. 2020;12:202\u0026ndash;207. doi:10.1007/s11930-020-00273-5.\u003c/li\u003e\n\u003cli\u003eBaud D, Pattaroni C, Vulliemoz N, Castella V, Marsland BJ, Stojanov M. Sperm microbiota and its impact on semen parameters. Front Microbiol. 2019;10:234. doi:10.3389/fmicb.2019.00234.\u003c/li\u003e\n\u003cli\u003eFarahani L, Tharakan T, Yap T, Ramsay JW, Jayasena CN, Minhas S. The semen microbiome and its impact on sperm function and male fertility: A systematic review and meta-analysis. Andrology. 2021;9:115\u0026ndash;144. doi:10.1111/andr.12886.\u003c/li\u003e\n\u003cli\u003eSchjenken JE, Robertson SA. The female response to seminal fluid. Physiol Rev. 2020;100:1077\u0026ndash;1117. doi:10.1152/physrev.00013.2018.\u003c/li\u003e\n\u003cli\u003eM\u0026auml;ndar R, Punab M, Borovkova N, Lapp E, Kiiker R, Korrovits P, et al. Complementary seminovaginal microbiome in couples. Res Microbiol. 2015;166:440\u0026ndash;447. doi:10.1016/j.resmic.2015.03.009.\u003c/li\u003e\n\u003cli\u003ePazgier M, Hoover DM, Yang D, Lu W, Lubkowski J. Human beta-defensins. Cell Mol Life Sci. 2006;63:1294\u0026ndash;1313. doi:10.1007/s00018-005-5540-2.\u003c/li\u003e\n\u003cli\u003eHolly MK, Diaz K, Smith JG. Defensins in viral infection and pathogenesis. Annu Rev Virol. 2017;4:369\u0026ndash;391. doi:10.1146/annurev-virology-101416-041734.\u003c/li\u003e\n\u003cli\u003eTollner TL, Venners SA, Hollox EJ, Yudin AI, Liu X, Tang G, et al. A common mutation in the defensin DEFB126 causes impaired sperm function and subfertility. Sci Transl Med. 2011;3:92ra65. doi:10.1126/scitranslmed.3002289.\u003c/li\u003e\n\u003cli\u003eDiao R, Fok KL, Chen H, Yu MK, Duan Y, Chung CM, et al. Deficient human \u0026beta;-defensin 1 underlies male infertility associated with poor sperm motility and genital tract infection. Sci Transl Med. 2014;6:249ra108. doi:10.1126/scitranslmed.3009071.\u003c/li\u003e\n\u003cli\u003eRadhakrishnan Y, Hamil KG, Yenugu S, Young SL, French FS, Hall SH. Identification, characterization, and evolution of a primate beta-defensin gene cluster. Genes Immun. 2005;6:203\u0026ndash;210. doi:10.1038/sj.gene.6364184.\u003c/li\u003e\n\u003cli\u003eWang M, Liu X, Chang G, Chen Y, An G, Yan L, et al. Single-Cell RNA Sequencing Analysis Reveals Sequential Cell Fate Transition during Human Spermatogenesis. Cell Stem Cell. 2018;23:599\u0026ndash;614.e4. doi:10.1016/j.stem.2018.08.007.\u003c/li\u003e\n\u003cli\u003eJin J, Li X, Ye M, Qiao F, Chen H, Fok KL. Defb19 regulates the migration of germ cell and is involved in male fertility. Cell Biosci. 2022;12:188. doi:10.1186/s13578-022-00924-1.\u003c/li\u003e\n\u003cli\u003eLi X, Yuan C, Shi J, Kang H, Chen Y, Duan Y, et al. \u0026beta;-Defensin 19/119 mediates sperm chemotaxis and is associated with idiopathic infertility. Cell Rep Med. 2022;:100825. doi:10.1016/j.xcrm.2022.100825.\u003c/li\u003e\n\u003cli\u003eWorld Health Organization. WHO laboratory manual for the examination and processing of human semen. 5th ed. Geneva: World Health Organization; 2010.\u003c/li\u003e\n\u003cli\u003eBolyen E, Rideout JR, Dillon MR, Bokulich NA, Abnet CC, Al-Ghalith GA, et al. Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. Nat Biotechnol. 2019;37:852\u0026ndash;857. doi:10.1038/s41587-019-0209-9.\u003c/li\u003e\n\u003cli\u003eChen S, Zhou Y, Chen Y, Gu J. fastp: an ultra-fast all-in-one FASTQ preprocessor. Bioinformatics. 2018;34:i884\u0026ndash;i890. doi:10.1093/bioinformatics/bty560.\u003c/li\u003e\n\u003cli\u003eCallahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581\u0026ndash;583. doi:10.1038/nmeth.3869.\u003c/li\u003e\n\u003cli\u003eCallahan BJ, McMurdie PJ, Holmes SP. Exact sequence variants should replace operational taxonomic units in marker-gene data analysis. ISME J. 2017;11:2639\u0026ndash;2643. doi:10.1038/ismej.2017.119.\u003c/li\u003e\n\u003cli\u003eDeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006;72:5069\u0026ndash;5072. doi:10.1128/AEM.03006-05.\u003c/li\u003e\n\u003cli\u003eLiu C, Cui Y, Li X, Yao M. microeco: an R package for data mining in microbial community ecology. FEMS Microbiol Ecol. 2021;97. doi:10.1093/femsec/fiaa255.\u003c/li\u003e\n\u003cli\u003eForster SC, Kumar N, Anonye BO, Almeida A, Viciani E, Stares MD, et al. A human gut bacterial genome and culture collection for improved metagenomic analyses. Nat Biotechnol. 2019;37:186\u0026ndash;192. doi:10.1038/s41587-018-0009-7.\u003c/li\u003e\n\u003cli\u003eYan Z, Chen B, Yang Y, Yi X, Wei M, Ecklu-Mensah G, et al. Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions. Nat Microbiol. 2022;7:1361\u0026ndash;1375. doi:10.1038/s41564-022-01196-8.\u003c/li\u003e\n\u003cli\u003eNakatsu G, Li X, Zhou H, Sheng J, Wong SH, Wu WKK, et al. Gut mucosal microbiome across stages of colorectal carcinogenesis. Nat Commun. 2015;6:8727. doi:10.1038/ncomms9727.\u003c/li\u003e\n\u003cli\u003eLangfelder P, Horvath S. WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics. 2008;9:559. doi:10.1186/1471-2105-9-559.\u003c/li\u003e\n\u003cli\u003eSchutte BC, McCray PB. [beta]-defensins in lung host defense. Annu Rev Physiol. 2002;64:709\u0026ndash;748. doi:10.1146/annurev.physiol.64.081501.134340.\u003c/li\u003e\n\u003cli\u003eFruitwala S, El-Naccache DW, Chang TL. Multifaceted immune functions of human defensins and underlying mechanisms. Semin Cell Dev Biol. 2019;88:163\u0026ndash;172. doi:10.1016/j.semcdb.2018.02.023.\u003c/li\u003e\n\u003cli\u003eYang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, et al. Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science. 1999;286:525\u0026ndash;528. doi:10.1126/science.286.5439.525.\u003c/li\u003e\n\u003cli\u003eSelsted ME, Ouellette AJ. Mammalian defensins in the antimicrobial immune response. Nat Immunol. 2005;6:551\u0026ndash;557. doi:10.1038/ni1206.\u003c/li\u003e\n\u003cli\u003eLi P, Chan HC, He B, So SC, Chung YW, Shang Q, et al. An antimicrobial peptide gene found in the male reproductive system of rats. Science. 2001;291:1783\u0026ndash;1785. doi:10.1126/science.1056545.\u003c/li\u003e\n\u003cli\u003eYenugu S, Hamil KG, Radhakrishnan Y, French FS, Hall SH. The androgen-regulated epididymal sperm-binding protein, human beta-defensin 118 (DEFB118) (formerly ESC42), is an antimicrobial beta-defensin. Endocrinology. 2004;145:3165\u0026ndash;3173. doi:10.1210/en.2003-1698.\u003c/li\u003e\n\u003cli\u003eContreras G, Shirdel I, Braun MS, Wink M. Defensins: Transcriptional regulation and function beyond antimicrobial activity. Dev Comp Immunol. 2020;104:103556. doi:10.1016/j.dci.2019.103556.\u003c/li\u003e\n\u003cli\u003eYang H, Zhang J, Xue Z, Zhao C, Lei L, Wen Y, et al. Potential Pathogenic Bacteria in Seminal Microbiota of Patients with Different Types of Dysspermatism. Sci Rep. 2020;10:6876. doi:10.1038/s41598-020-63787-x.\u003c/li\u003e\n\u003cli\u003eMores CR, Price TK, Wolff B, Halverson T, Limeira R, Brubaker L, et al. Genomic relatedness and clinical significance of Streptococcus mitis strains isolated from the urogenital tract of sexual partners. Microb Genom. 2021;7. doi:10.1099/mgen.0.000535.\u003c/li\u003e\n\u003cli\u003eMoretti E, Capitani S, Figura N, Pammolli A, Federico MG, Giannerini V, et al. The presence of bacteria species in semen and sperm quality. J Assist Reprod Genet. 2009;26:47\u0026ndash;56. doi:10.1007/s10815-008-9283-5.\u003c/li\u003e\n\u003cli\u003eWeng S-L, Chiu C-M, Lin F-M, Huang W-C, Liang C, Yang T, et al. Bacterial communities in semen from men of infertile couples: metagenomic sequencing reveals relationships of seminal microbiota to semen quality. PLoS One. 2014;9:e110152. doi:10.1371/journal.pone.0110152.\u003c/li\u003e\n\u003cli\u003eYeoh YK, Sun Y, Ip LYT, Wang L, Chan FKL, Miao Y, et al. Prevotella species in the human gut is primarily comprised of Prevotella copri, Prevotella stercorea and related lineages. Sci Rep. 2022;12:9055. doi:10.1038/s41598-022-12721-4.\u003c/li\u003e\n\u003cli\u003eWhiley RA, Beighton D, Winstanley TG, Fraser HY, Hardie JM. Streptococcus intermedius, Streptococcus constellatus, and Streptococcus anginosus (the Streptococcus milleri group): association with different body sites and clinical infections. J Clin Microbiol. 1992;30:243\u0026ndash;244. doi:10.1128/jcm.30.1.243-244.1992.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"β-defensin, bacterial networks, host-microbe interaction, microbiota","lastPublishedDoi":"10.21203/rs.3.rs-2716767/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2716767/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInfertility is associated with the alteration of the seminal microbiome. However, how the onset of dysbiosis remains controversial and the involvement of host factors remains elusive. This study investigates the alterations of the seminal microbiome in male infertility and examines the association and function of DEFB119, a reproductive-tract-specific host antimicrobial peptide, on the seminal microbiome and male fertility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe analyzed the seminal microbiome by 16S rRNA sequencing in 30 individuals with normal sperm parameters and 58 male-factor infertile patients. While we observed comparable genera, diversity and evenness of bacterial communities, a marked decrease in the modularity of the metacommunities was observed in patients with abnormal spermiogram. Analysis of the protein level of DEFB119 by ELISA revealed a marked elevation of DEFB119 in a subpopulation of male-infertile patients. Elevated seminal DEFB119 was associated with a decrease in the observed genera, diversity and evenness of bacterial communities and further distortion of the metacommunities. Mediation analysis suggests the involvement of elevated DEFB119 and dysbiosis of the seminal microbiome in mediating the abnormalities in the spermiogram. Functional experiments showed that recombinant DEFB119 significantly decrease the progressive motility of sperm in patients with abnormal spermiogram. Moreover, DEFB119 demonstrated species-specific antimicrobial activity against common seminal and non-seminal species.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMale infertility is associated with distorted bacterial networks. Elevation of the DEFB119 level in seminal plasma stratifies male infertile patients with dysbiosis of the seminal microbiome. Both elevated DEFB119 and dysbiosis contribute to the abnormal spermiogram. Our work identifies an important host factor that mediates the host-microbiome interaction and stratifies the seminal microbiome associated with male infertility. These results may lead to a new diagnostic method for male infertility and regimens for formulating the microbiome in the reproductive tract and other organ systems.\u003c/p\u003e","manuscriptTitle":"DEFB119 stratifies dysbiosis with distorted networks in the seminal microbiome associated with male infertility","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-26 20:03:59","doi":"10.21203/rs.3.rs-2716767/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"7b3b52ad-f92a-478b-a17c-f830b32275d3","owner":[],"postedDate":"April 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-06-05T17:44:17+00:00","versionOfRecord":[],"versionCreatedAt":"2023-04-26 20:03:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2716767","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2716767","identity":"rs-2716767","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}